Project Summary/Abstract This project is focused on the mechanisms involved in human maternal-fetal tolerance, an immunological enigma described by Peter Medawar more than 50 years ago. The fetus in utero bears paternal antigens and should be rejected as a foreign graft by the maternal immune system. Multiple mechanisms contribute to implantation tolerance. Maternal leukocytes that populate the uterine decidua certainly play an important role. They interact with placental trophoblasts that attach to and invade the decidua (implantation) and that express an unusual Class I MHC protein of unknown function, the HLA-G dimer, that is found only in pregnant women and apes. The goal of this proposal is to characterize maternal leukocytes and fetal trophoblasts and to study their interaction that must contribute to various aspects of pregnancy, including remodeling of the uterine spiral arteries to increase blood flow to the developing fetus, implantation of the blastocyst (16 cell stage embryo) into the uterus and maternal-fetal tolerance. Decidual natural killer cells (50-90% of leukocytes in the decidua that were characterized during the first 5 years of this project) contribute to maternal-fetal tolerance by several means. This proposal seeks to expand our knowledge of the interaction of HLA-G dimer bearing cells (initially using an HLA-G+ transfected cell line) with decidual Natural Killer cells and macrophages, to describe synapses that occur between these cells, and to characterize the second most populous set of decidual leukocytes, the macrophages (20% of the leukocytes). In addition to inflammatory macrophages (M1) that engulf and dispose of noxious materials such as bacteria, a second type of macrophage (M2) has been described in mice that have an immunoregulatory role. Microarray analysis will be used as a principal tool in characterizing human decidual macrophages in order to establish to which subtype they belong and what factors they may secrete. We hypothesize that they are M2 macrophages. A further goal is to isolate and characterize HLA-G+ extravillous trophoblasts that invade the decidua and intermingle with the decidual leukocytes in order to study directly the interaction of HLA-G+ trophoblasts with decidual natural killer cells and macrophages. This interaction may be different from the interaction of the leukocytes with HLA-G transfectants. We have already established that these interactions lead to the secretion of several cytokines and hypothesize that other proteins involved in maternal-fetal tolerance and in vascular remodeling are also secreted. Finally, the mechanism by which LIF produced by decidualized uterine epithelium and/or macrophages contributes to the implantation process will be examined by studying its effects on trophoblasts that initiate this process. It has been reported that LIF knockout female mice are infertile because the blastocyst formed after conception cannot implant into the uterus. This project is directly related to reproductive failure in women, i.e. recurrent spontaneous abortion (RSA), some forms of infertility due to failure of implantation of the blastocyst, preeclampsia and fetal growth restriction due to the failure of vascular remodeling that provides an adequate blood supply.